Name: alveolar macrophage phagocytosis and bacteria clearance in mice
Uploaded: 2019-03-02T14:00:00
Description: Watch this Scientific Journal Video about Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice at JoVE.com

这项工作得到 r01hl116826 至 x. zhao 的支持。

该协议遵循东弗吉尼亚医学院动物护理和使用机构委员会 (iacuc) 的指导原则。
1. 荧光珠吞噬作用
<ol><li>根据 iacuc 对动物的伦理安乐死协议, 用 co2 窒息对小鼠进行安乐死 (c57bl/6j, 6周大, 女性)。</li>
	<li>将鼠标放在覆盖有纸巾的解剖板上。用爪子把它的爪子固定下来, 四肢展开老鹰, 在门牙下钩住一根绳子, 将它的头向后拉, 这样气管就被垂直和水平地定位。</li>
	<li>用7...

<ol>
	<li>Hussell, T., Bell, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages:+plasticity+in+a+tissue-specific+context.">Alveolar macrophages: plasticity in a tissue-specific context.</a> Nature Reviews Immunology. 14, 81-93 (2014).</li><li>Belchamber, K. B. R., Donnelly, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+Dysfunction+in+Respiratory+Disease.">Macrophage Dysfunction in Respiratory Disease.</a> Results and Problems in Cell Differentiation. 62, 299-313 (2017).</li><li>Broug-Holub, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages+are+required+for+protective+pulmonary+defenses+in+murine+Klebsiella+pneumonia:+elimination+of+alveolar+macrophages+increases+neutrophil+recruitment+but+decreases+bacterial+clearance+and+survival.">Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival.</a> Infection and Immunity. 65, 1139-1146 (1997).</li><li>Knapp, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages+have+a+protective+antiinflammatory+role+during+murine+pneumococcal+pneumonia.">Alveolar macrophages have a protective antiinflammatory role during murine pneumococcal pneumonia.</a> American Journal of Respiratory and Critical Care Medicine. 167, 171-179 (2003).</li><li>Bhatia, M., Zemans, R. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1beta+regulates+CXCL8+release+and+influences+disease+outcome+in+response+to+Streptococcus+pneumoniae,+defining+intercellular+cooperation+between+pulmonary+epithelial+cells+and+macrophages.">Interleukin-1beta regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages.</a> Infection and Immunity. 80, 1140-1149 (2012).</li><li>Greenlee-Wacker, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+of+apoptotic+neutrophils+and+resolution+of+inflammation.">Clearance of apoptotic neutrophils and resolution of inflammation.</a> Immunological Reviews. 273, 357-370 (2016).</li><li>Haslett, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte+apoptosis+and+its+role+in+the+resolution+and+control+of+lung+inflammation.">Granulocyte apoptosis and its role in the resolution and control of lung inflammation.</a> American Journal of Respiratory and Critical Care Medicine. 160, 5-11 (1999).</li><li>Cox, G., Crossley, J., Xing, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+engulfment+of+apoptotic+neutrophils+contributes+to+the+resolution+of+acute+pulmonary+inflammation+in+vivo.">Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo.</a> American Journal of Respiratory Cell and Molecular Biology. 12, 232-237 (1995).</li><li>Groves, E., Dart, A. E., Covarelli, V., Caron, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+phagocytic+uptake+in+mammalian+cells.">Molecular mechanisms of phagocytic uptake in mammalian cells.</a> Cellular and Molecular Life Sciences. 65, 1957-1976 (2008).</li><li>Mosser, D. M., Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Opsonic+Phagocytosis+via+Fc&#947;+Receptors+and+complement+receptors+on+macrophages.">Measuring Opsonic Phagocytosis via Fc&#947; Receptors and complement receptors on macrophages.</a> Current Protocols in Immunology. , (2011).</li><li>He, J. Q., Wiesmann, C., van Lookeren Campagne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+of+macrophage+complement+receptor+CRIg+in+immune+clearance+and+inflammation.">A role of macrophage complement receptor CRIg in immune clearance and inflammation.</a> Molecular Immunology. 45, 4041-4047 (2008).</li><li>Nagre, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Macrophage+Complement+Receptor+CRIg+by+TRIM72+Polarizes+Innate+Immunity+of+the+Lung.">Inhibition of Macrophage Complement Receptor CRIg by TRIM72 Polarizes Innate Immunity of the Lung.</a> American Journal of Respiratory Cell and Molecular Biology. 58 (6), 756-766 (2018).</li><li>Miksa, M., Komura, H., Wu, R., Shah, K. G., Wang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Method+to+Determine+the+Engulfment+of+Apoptotic+Cells+by+Macrophages+using+pHrodo+Succinimidyl+Ester.">A Novel Method to Determine the Engulfment of Apoptotic Cells by Macrophages using pHrodo Succinimidyl Ester.</a> Journal of Immunological Methods. 342 (1-2), 71-77 (2009).</li><li>Su, H., Chen, H., Jen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+exercise+enhances+phagocytosis+by+murine+bronchoalveolar+macrophages.">Severe exercise enhances phagocytosis by murine bronchoalveolar macrophages.</a> Journal of Leukocyte Biology. 69, 75-80 (2001).</li><li>Amiel, E., Lovewell, R. R., O'Toole, G. A., Hogan, D. A., Berwin, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa.+evasion+of+phagocytosis+is+mediated+by+loss+of+swimming+motility+and+is+independent+of+flagellum+expression.">Pseudomonas aeruginosa. evasion of phagocytosis is mediated by loss of swimming motility and is independent of flagellum expression.</a> Infection and Immunity. 78, 2937-2945 (2010).</li><li>Giannoni, E., Sawa, T., Allen, L., Wiener-Kronish, J., Hawgood, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactant+Proteins+A+and+D+Enhance+Pulmonary+Clearance+of+Pseudomonas+aeruginosa.">Surfactant Proteins A and D Enhance Pulmonary Clearance of Pseudomonas aeruginosa.</a> American Journal of Respiratory Cell and Molecular Biology. 34, 704-710 (2006).</li></ol>

在发挥气体交换功能的同时, 肺持续面对外来颗粒、病原体和过敏原。ams 凭借其主要功能, 即吞噬作用, 提供了第一道防线。ams 还与其他免疫细胞协调, 以摧毁病原体和解决炎症。在这里, 我们描述了从小鼠肺分离的 ams 专门评估吞噬性的方法。本手稿中提出的协议解释了在体内和体外对吞噬作用的详细研究, 该研究也可用于研究其他器官的巨噬细胞功能和细菌清除。
所描述的?...

ams 是肺泡中处于休眠阶段的主要脂肪细胞, 也是通过识别和内化吸入病原体和外来颗粒 1, 2 而产生先天免疫反应的主要参与者之一。据报道, ams 对于快速清除铜绿假单胞菌和3,4 肺炎克雷伯菌等多种肺部病原体至关重要, 因此 am 吞噬能力的缺乏往往导致呼吸道疾病。感染, 如急性肺炎, 导致较高的死亡率和发病率。
ams 还通过产生细胞因子和趋化因子 (如 tnf-α和 il-1β) 在肺中引发先天炎症反应, 这些细胞与肺泡环境中的其他细胞相通, 产生趋化因子并诱导炎症中性粒细胞、单核细胞和自适应免疫细胞在肺5。例如, ams 产生的 il-1β有助于促进中性粒细胞趋化因子 cxcl8 从上皮细胞6中释放。此外, ams 还有助于凋亡多核白细胞 (pmn) 的吞噬, 而这种白细胞的失败会导致细胞内酶从 pmn 持续泄漏到周围组织, 从而导致组织损伤和长期炎症。7.,8,9个。
ams 的吞噬作用是通过对病原体表面病原体相关分子模式的直接识别, 通过 ams 的模式识别受体 (prr) 或通过将凋亡的病原体与 ams 的免疫效应受体结合而介导的10. 对于后者, ams 可以通过其 fcγ受体 (fcγr) 识别用免疫球蛋白 (igg) 进行光声的目标, 或通过其补体受体 (cr)11识别含有补体片段 c3b 和 c3bi 的病原体。在补体受体中, 免疫球蛋白超家族 (crig) 的 cr 在组织巨噬细胞12中选择性表达, 最近的一项发现强调了 crig 在铜绿假单胞菌肺炎背景下的作用。13岁
许多原始的研究使用的方法来评估巨噬细胞吞噬, 以描述巨噬细胞功能14,15 的分子机制。然而, 像体内吞噬作用这样的方法需要精确的吞噬量的定量。本文综述了用荧光素异硫氰酸酯 (fitc) 玻璃珠和铜绿绿绿色荧光蛋白 (gfp) 进行体外和体内吞噬的详细方法。此外, 我们还解释了在 prr-、cr-r 和 fcγ r 介导的吞噬作用之间的鉴别方法。最后, 我们报告了一种方法来描述细菌清除在小鼠与铜绿假单胞菌肺炎。

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			<td height="21" style="height:21px;width:216px;">18-G Needle</td>
			<td style="width:179px;">Nipro Medical&#160;</td>
			<td style="width:143px;">CI+1832-2C</td>
			<td style="width:272px;">Molecular Biology grade</td>
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			<td height="21" style="height:21px;width:216px;">2,7-diaminofluorene (DAF)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">D17106</td>
			<td>Molecular Biology grade</td>
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			<td height="21" style="height:21px;width:216px;">70% Ethanol</td>
			<td style="width:179px;">Decon Labs Inc.</td>
			<td style="width:143px;">18C27B</td>
			<td>Analytical grade</td>
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			<td style="width:179px;">Corning</td>
			<td style="width:143px;">3603</td>
			<td>Cell Biology grade</td>
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			<td style="width:179px;">Life Technologies</td>
			<td style="width:143px;">A10492</td>
			<td>Molecular Biology grade</td>
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			<td height="42" style="height:42px;width:216px;">Alexa fluor-488 Zymosan-A-bioparticle</td>
			<td style="width:179px;">Thermofisher Scientific</td>
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			<td>Molecular Biology grade</td>
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			<td style="width:179px;">Sigma-Aldrich</td>
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			<td>Biochemical reagent</td>
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			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
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			<td></td>
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			<td style="width:143px;">11995-065</td>
			<td>Cell Biology grade</td>
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			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:179px;">Atlanta Biologicals</td>
			<td style="width:143px;">S11550</td>
			<td>Cell Biology grade</td>
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			<td height="21" style="height:21px;width:216px;">Flow Cytometer</td>
			<td style="width:179px;">BD Biosciences</td>
			<td style="width:143px;">FACSCalibur</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Flow Jo Software</td>
			<td style="width:179px;">FlowJo, LLC</td>
			<td style="width:143px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:179px;">Dumont</td>
			<td style="width:143px;">0508-SS/45-PS-1</td>
			<td>Suitable for laboratory animal dissection</td>
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			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">L4530</td>
			<td>Cell Biology grade</td>
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			<td height="21" style="height:21px;width:216px;">GFP-P. aeruginosa</td>
			<td style="width:179px;">ATCC</td>
			<td>101045GFP</td>
			<td>Suitable for&#160; cell infection assays</td>
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			<td height="21" style="height:21px;width:216px;">Glass bottom dish</td>
			<td style="width:179px;">MatTek Corp.</td>
			<td>P35G-0.170-14-C</td>
			<td>Cell Biology grade</td>
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			<td height="21" style="height:21px;width:216px;">High-Pressure Syringe</td>
			<td style="width:179px;">Penn-Century</td>
			<td>FMJ-250</td>
			<td>Suitable for laboratory animal use</td>
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			<td height="21" style="height:21px;width:216px;">Homogenizer</td>
			<td style="width:179px;">Omni International</td>
			<td>TH-01</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Hydrogen peroxide</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">H1009</td>
			<td>Analytical grade</td>
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			<td height="42" style="height:42px;width:216px;">Inverted Fluorescence Microscope</td>
			<td style="width:179px;">Olympus</td>
			<td style="width:143px;">IX73</td>
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			<td height="21" style="height:21px;width:216px;">Ketamine Hydrochloride</td>
			<td style="width:179px;">Hospira</td>
			<td style="width:143px;">CA-2904</td>
			<td>Pharmaceutical grade</td>
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			<td height="21" style="height:21px;width:216px;">Shandon Kwik-Diff Stains</td>
			<td style="width:179px;">Thermofisher Scientific</td>
			<td style="width:143px;">9990700</td>
			<td>Cell Biology grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB Agar</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:143px;">BP1425</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB Broth</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:143px;">BP1427</td>
			<td>Molecular Biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MicroSprayer Aerosolizer</td>
			<td style="width:179px;">Penn-Century</td>
			<td style="width:143px;">IA-1C</td>
			<td>Suitable for laboratory animal use</td>
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			<td height="21" style="height:21px;width:216px;">Paraformaldehyde</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">P6148</td>
			<td>Reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:179px;">Gibco</td>
			<td style="width:143px;">20012-027</td>
			<td>Cell Biology grade</td>
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			<td height="21" style="height:21px;">rabbit anti-SRBC-IgG&#160;</td>
			<td style="width:179px;">MP Biomedicals</td>
			<td style="width:143px;">55806</td>
			<td>Suitable for immuno-assays</td>
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			<td height="21" style="height:21px;">rabbit anti-SRBC-IgM&#160;</td>
			<td style="width:179px;">Cedarline Laboratories</td>
			<td style="width:143px;">CL9000-M</td>
			<td>Suitable for immuno-assays</td>
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			<td height="21" style="height:21px;">Scissors</td>
			<td style="width:179px;">Miltex</td>
			<td style="width:143px;">5-2</td>
			<td>Suitable for laboratory animal dissection</td>
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			<td style="width:179px;">Penn-Century</td>
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			<td>Suitable for laboratory animal use</td>
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			<td style="width:179px;">BioRad</td>
			<td style="width:143px;">1610301</td>
			<td>Analytical grade</td>
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		<tr height="21">
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			<td style="width:179px;">Fine Science Tools</td>
			<td style="width:143px;">15012-12</td>
			<td>Suitable for laboratory animal dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring Scissors (Small)</td>
			<td style="width:179px;">Fine Science Tools</td>
			<td style="width:143px;">91500-09</td>
			<td>Suitable for laboratory animal dissection</td>
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			<td height="21" style="height:21px;">sheep red blood cells (SRBCs)&#160;</td>
			<td style="width:179px;">MP Biomedicals</td>
			<td style="width:143px;">55876</td>
			<td>Washed, preserved SRBCs</td>
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			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:143px;">U5378</td>
			<td>Molecular Biology grade</td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">Xylazine&#160;</td>
			<td style="width:179px;">Akorn Animal Health</td>
			<td style="width:143px;">59399-110-20</td>
			<td>Pharmaceutical grade</td>
		</tr>
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</table>

alveolar macrophage phagocytosis and bacteria clearance in mice

肺泡巨噬细胞 (ams) 保护肺的肺泡空间。ams 的吞噬作用在防御入侵病原体、去除死细胞或异物以及解决炎症反应和组织重塑方面发挥着至关重要的作用, 这些过程是由各种表面受体介导的。ams。在这里, 我们报告了分析 ams 的吞噬功能的方法, 使用体外和体内的检测和实验策略, 以区分模式识别受体-, 补体受体, 和 fc 伽玛受体介导吞噬。最后, 我们讨论了建立和表征小鼠铜绿假单胞菌肺炎模型的方法, 以评估细菌在体内的清除率。这些检测是评估 am 功能最常用的方法, 也可用于研究巨噬细胞功能和其他器官的细菌清除。

我们首先进行了小鼠原发性 ams 吞噬分析的实验。在所有分析中, 我们比较了从 wt 和 trim72 ko 小鼠中分离出的 ams。如图 1a 所示, 荧光显微镜显示, 小鼠原代 ams 在孵育1小时后, 会发生 fitc-玻璃微珠的吞噬作用。图 1b显示流式细胞仪对吞噬性的分析。用显微镜和流式细胞仪测量的吞噬量的定量分别?...

Watch this Scientific Journal Video about Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice at JoVE.com

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

肺泡巨噬细胞吞噬作用与小鼠细菌清除

本文报道了分析小鼠肺泡巨噬细胞吞噬功能和肺细菌清除的常用方法。这些方法研究荧光素异硫氰酸珠的体外吞噬和铜绿假单胞菌绿色荧光蛋白的体内吞噬作用。我们还描述了一种清除小鼠铜绿假单胞菌的方法。

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

伪多多尼亚是一种BSL-2级病原体。在处理此生物体时，请记住遵循所有 BSL-2 级别安全实践。在这里，我们将演示原发细胞分离、体外噬菌体病和噬菌体通路分析，以及小鼠体内噬菌体和细菌清除评估。成功的腹内输送需要实践来掌握。确保微音器正确加载，针头位于两个声褶之间，柱塞速度得到适当控制。首先将鼠标固定在超活位置，四肢铺在用纸巾覆盖的解剖板上，并在门牙下钩上一根绳子来缩回头部，使气管定位直，水平。用70%乙醇对鼠标进行消毒，并使用常规钳子拉起身体中心线的皮肤。使用手术剪刀将皮肤从腹部切到喉咙顶部。使用标准手术剪刀的钝端仔细解剖喉部肌肉和结缔组织。打开肋骨下方的腹壁。切开隔膜，切掉肋骨的下部，部分暴露肺部。使用弹簧剪刀露出气管，并使用钳子抓住软骨环。使用微型剪刀小心地在气管的腹孔面上切开大约 1.5 毫米的切口。小心地将短长度的缝合线串在气管下面，并将一个 18 表管插入气管。当管管就位时，每次洗涤用一毫升新鲜 PBS 轻轻洗刷肺部三次，轻轻地将液体提取到注射器中，然后连续三次重新注入肺部。每次熔岩后，将收集的支气管熔岩液或BALF转移到15毫升锥形管中，以离心总收获的流体。将颗粒重新放入一毫升新鲜PBS中进行第二次离心，然后将洗涤的道利巨噬细胞重新泵入Dulbeco的改性鹰中或DMEM的两毫升中，辅以10%的非热灭活胎儿牛血清。然后将原发性道外巨噬细胞转移到玻璃底培养皿中，在37摄氏度的细胞培养箱中进行为期两天的培养。48小时后，用一毫升PBS清洗培养，然后用两毫升新鲜介质喂养培养。接下来，将每个细胞的50个直径为2微米直径的箱式乳胶进发乳胶、结合珠子加入培养，在细胞培养箱中以37摄氏度的温度下孵育一小时。在孵育结束时，每次洗涤用一毫升新鲜PBS广泛清洗板五次，取出细胞外珠，并随机成像100个细胞，以计算含有细胞内珠的细胞。对于结果化测定，在室温下用50微升的兔子抗SRBC免疫球蛋白M或IgM孵育2倍10至第八羊红血球或SRBCs，在室温下孵育30分钟。然后在37摄氏度下用50微升C5缺乏的人类血清孵育蛋白化SRBCs，在37摄氏度下孵育30分钟，以修复IgM涂层SRBCs上的C3b和C3b抑制剂补充片段。接下来，吸气介质，将 100 微升（1 倍 10）添加到每毫升介质的 7 个反化 SRBC 中，每 96 井板包含 1 倍 10 至第四个通宵培养的 murine 巨噬细胞。在37摄氏度下孵育SRB小鼠巨噬细胞共培养一小时，然后用100微升氯化铵解钾解液缓冲液清洗，取出未绑定的SRBCs。移除未绑定的 SRBC 后，使用 100 微升介质冲洗。用0.1%的硫酸钠将剩余的细胞酶化，用50微升的2、7-二甲基黄丙烯，辅以3%过氧化氢和6个摩尔尿素来治疗赖酸盐。然后在620纳米的分光光度计上测量血红蛋白催化氟烯蓝色地层吸收度。使用具有已知 SRBC 数量的 620 纳米吸收度值的标准曲线来确定邻苯二甲酸化 SRBC 的数量。为了评估模式识别受体中位噬菌体，用1毫升PBS清洗两天培养小鼠原肺泡巨噬细胞，用含有100个Alexa氟-488结合的Zymosan-A-生物粒子的500微升新鲜介质治疗细胞。在37摄氏度下一小时后，用500微升的冰冷PBS来阻止吞噬，每次洗涤一毫升PBS，对细胞进行五次大面积清洗。上次洗涤后，在室温下用4%甲醛固定细胞10分钟，然后像证明的另外5次清洗细胞。上次洗涤后，用500微升新鲜PBS覆盖细胞，在差分干扰对比度和荧光通道下以488纳米进行成像，以量化含有齐莫桑-A-生物粒子的道白道巨噬细胞的数量。对于体内肺囊巨噬细胞位评估，确认适当的水平的电合，在麻醉小鼠的手指捏没有反应，并放置鼠标在平板与塑料线下上切口。将鼠标放在半卧式讲台上，位置为 45 度，心室朝上，使用弯曲钳子拉出并抑制舌头。然后插入一个微普拉耶之间的声乐褶皱，以内管内管理50微升五倍10到6个殖民地形成单位的P.aeruginosa GFP到麻醉动物的肺部。要确认微普拉耶针在气管中，请轻轻移动注射器并观察针头两侧的声褶。感染一小时后，收集熔岩液，正如刚刚证明的，通过离心分解对起源的巨噬细胞进行颗粒化。将10倍的三分之一的新鲜PBS重新淹没在玻璃幻灯片上，用于细胞离心。然后根据标准组织化学方案，对道拉巨噬细胞、嗜中性粒细胞和淋巴细胞进行分色染色。随机选择100个道道巨噬细胞，以量化噬菌体的细胞百分比。在第一次体内细菌清除试验中，在两节内将每毫升P.aeruginosa第五菌群形成单位约2.5至5倍的亚莱特剂量注射到麻醉野生型和突变小鼠中，并在六天内每天测量每只动物的体重。在第二次试验中，将一组新的野生型小鼠和突变小鼠注射至每毫升剂量的7个菌落形成单位，并在注射后两天内记录死亡率，在注射死亡时或注射后两天内收集整个肺组织，以在高峰感染时量化肺细菌负担。收获肺部后，将肺样本切成小块，使用调整后的电动均质器设置使肺碎片均质。然后板100微升的均质到伪多莫纳斯隔离加糖板在10倍的连续稀释。荧光显微镜显示，小鼠初级肺功能巨噬细胞噬菌体感染的 FITC 乳胶珠发生在一个小时的孵育后，与 TRIM72 敲除巨噬细胞显示明显更高的噬菌体能力。相反，TRIM72（一种在巨噬细胞中功能不明的蛋白质）的过度表达，导致补充吞噬症减少五倍以上。然而，亚历克萨氟-488结合的Zymosan-A粒子被从野生型或敲除小鼠中分离出的主要道长巨噬细胞以相等数量摄入。从野生类型和淘汰动物收获的BALF的差别染色揭示了类似数量的巨噬细胞，但从敲除小鼠中收获的巨噬细胞的噬菌体能力更高。此外，TRIM72淘汰小鼠表现出更快的恢复其体重，保持其生存，并表现出比野生类型动物在内切内P.aeruginosa管理后较低的细菌负担。利用这项技术，可以分析其他对肺炎重要的噬菌体过程，如嗜中性粒细胞病和其他噬细胞在细菌清除中的相对贡献。结合药理抑制剂、适应性转移和转基因动物，该技术可以帮助研究人员探索特定类型的噬菌体分子成分。

alveolar-macrophage-phagocytosis-and-bacteria-clearance-in-mice

Research

JoVE Journal

Immunology and Infection

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

科研

免疫与感染

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

巨噬细胞胆固醇消耗及其对的吞噬作用<em&gt;新型隐球菌</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

细胞凋亡细胞的吞噬作用<em&gt;果蝇</em&gt;胚胎

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

增强型绿色荧光蛋白表达大肠杆菌评价小鼠腹腔巨噬细胞吞噬细胞增多

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

Assaying for Inorganic Polyphosphate in Bacteria

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species.

细菌中无机聚磷酸盐的检测

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction.

用于阿尔韦拉尔巨噬细胞和CD4和T细胞免疫造血和HIV储液评估的支气管性洗浴液和匹配血液的处理

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing ...

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

在臭氧暴露后阿尔韦拉巨噬细胞的Vivo评估中

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

利用流细胞测定人类白细胞测定阿斯珀吉鲁斯紫锥体烟曲的噬菌体

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...